Use of anti-embryonic hemoglobin antibodies to identify fetal cells

ABSTRACT

An in vitro method of identifying or isolating fetal cells from a blood sample is described. Fetal nucleated erythrocytes or erythroblasts are identified by using an antibody or antibody fragment specific for embryonic hemoglobin or an embryonic hemoglobin chain. Once the fetal cells are identified, they can be treated to render the fetal nucleic acids or proteins available for identification or amplification. Detecting the occurrence or existence of selected fetal nucleic acids or proteins allows a quantitative or qualitative diagnostic or prenatal evaluation, including determining the sex of the fetus, determining chromosomal, single gene or protein abnormalities, and determining the presence or absence of particular genes, nucleic acid sequences or proteins.

This invention relates to a method for separating and recognizing fetalcells from a blood sample. More particularly, it relates to theisolation and recognition of fetal nucleated erythrocytes orerythroblasts from maternal cells in a blood sample from a pregnantwoman.

BACKGROUND OF THE INVENTION

Fetal tissue, and in particular fetal DNA and chromosomes, is routinelyused in prenatal diagnosis and other medical procedures that require anaccurate assessment of the genome of the fetus. Currently, the fetaltissue is obtained by the use of amniocentesis, chorionic villussampling (CVS), fetoscopy, or cordocentesis, as described in Thompsonand Thompson Genetics in Medicine, 5th Edition, W. B. Saunders Co.,Philadelphia, 1991.

In amniocentesis, a sample of amniotic fluid, which contains fetalcells, is transabdominally removed from the mother with a needle andsyringe. Amniocentesis has inherent associated risks. The major risk isinduction of miscarriage which is estimated to occur in 1 in 200amniocenteses. Other risks include maternal infection and physicaldamage to the fetus. In CVS, trophoblast tissue is aspirated from thevillous area of the chorion transcervically or transabdominally. Therate of fetal loss by this method may be as high as 1 in 100.Cordocentesis or percutaneous umbilical blood sampling provides a methodof obtaining fetal blood directly from the umbilical cord withultrasonic guidance. Each of these invasive methods carries risks toboth the mother and the fetus.

Accordingly, it would be desirable to have a non-invasive method forobtaining fetal tissue or fetal DNA. It would also be desirable to havea method that is rapid and reliable for isolating and enriching thefetal tissue from maternal tissue in order to facilitate screening andpre-natal diagnosis in clinical laboratories. Recently, the preferredmethodology has been the identification of fetal cells in the peripheralmaternal circulation and then garnering of those cells for geneticanalysis.

Identification or isolation of fetal cells from maternal blood hasrelied upon distinguishing the rare population of fetal cells from themore prevalent maternal cells. Although various fetal cell types, suchas fetal lymphocytes and trophoblasts, have been utilized in theidentification process as target cells for fetal DNA, more efforts havebeen directed to fetal nucleated red blood cells (nRBC), also known asnucleated erythrocytes. See Cheuh and Golbus, "The Search for FetalCells in the Maternal Circulation", J. Perinatol. Med., 19:411 (1991);Simpson, et al., "Noninvasive Screening for Prenatal Genetic Diagnosis",Bull. WHO, 73:799 (1995); and Cheuh and Golbus, "Prenatal DiagnosisUsing Fetal Cells from Maternal Circulation", West. J. Med., 159(3):308(1993).

Fetal RBCs are thought to cross the placenta as a result oftransplacental bleeding. Since the fetus has a large number of nucleatederythrocytes, which nucleated erythrocytes are rarely found in adultblood, the difference in nucleation is useful in separating andidentifying fetal cells from maternal ones.

Antibodies to cell surface antigens particular to nRBCs, such as thetransferrin receptor, have been utilized to identify and enrich forthese fetal cells. See Bianchi, et al., "Isolation of Fetal DNA fromNucleated Erythrocytes in Maternal Blood", Proc. Natl. Acad. Sci.,87:3279 (1990). See also Bianchi, et al. PCT International ApplicationNo. PCT/US90/06623 (WO 91/07660), which describes a method for enrichingfetal nucleated red blood cells from a peripheral blood sample by theuse of an antibody which binds an antigen on the cell surface of thefetal cells.

Bresser, et al., PCT International Application No. PCT/US94/08342 (WO95/03431) describes the use of fetal hemoglobin antibodies and mRNAprobes to enrich for fetal cells in maternal blood. The presence offetal hemoglobin also has been demonstrated by the Kleihauer-Betkereaction that differentiates fetal from adult hemoglobin by acid elutioncharacteristics. See Kleihauer, et al., "Demonstration yon fetalemhamoglobin in den erythrocyten eines blutausstrichs", Klin. Woschenschr,35:637 (1957); and Saunders, et al., "Enrichment of fetal cells frommaternal blood for genetic analysis", American Journal of HumanGenetics, 57:287 (1995).

Genetic analysis of the fetal genome has been accomplished byfluorescence in situ hybridization (FISH) of chromosome or gene specificDNA or RNA probes, sometimes with automated reading, and byamplification of targeted fetal genes or DNA. See Lichter, et al.,"Rapid detection of human chromosome 21 aberration analysis usingfluorescence in situ hybridization", Proc. Natl. Acad. Sci., 85:9664(1988); O'Kelley, et al., "Instrumentation for the genetic evaluation offetal cells from maternal blood", Am. J. Hum. Genet., 57:286 (1995); andLo, et al., "Prenatal Sex Determination by DNA amplification frommaternal peripheral blood", Lancet, 2:1363 (1989).

SUMMARY OF THE INVENTION

The present invention provides a method of identifying a fetalerythrocyte, preferably nucleated, or an erythroblast cell in a bloodsample, the method comprising

a) contacting the blood sample with an antibody, or antibody fragmentthereof, directed to an embryonic globin portion of hemoglobin, whereinthe antibody or fragment will bind the fetal cell; and

b) identifying the cells which bind to the antibody or fragment as fetalnucleated erythrocyte or erythroblast cells.

(The blood sample is typically taken from the peripheral circulatingmaternal blood during gestation.)

Various anti-embryonic hemoglobin antibodies or antibody fragmentsthereof can be used in the method, preferably those directed to theembryonic epsilon globin chain and/or embryonic zeta globin chain ofhemoglobin. In addition, second fetal or mature cell markers can beutilized to further identify or isolated the desired fetal cell.

Once the selected fetal cells are identified, a fetal nucleic acid orprotein can be amplified or detected within the cell for geneticanalysis.

Also provided by the invention are various kits for use in conjunctionwith the methods described above. These kits comprise a directly orindirectly labeled anti-embryonic hemoglobin antibody and instructionsfor use. Also optionally included within such kits are density gradientmediums for enriching the concentration of the fetal cells, hemolysisreagents for disrupting red blood cells, lysis agents, and nucleic acidprobes.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

As used herein, "erythrocytes" or "red blood cells" or "RBC" includeadult and fetal red blood cells, and may be nucleated or non-nucleated.Nucleated erythrocytes are preferred.

As used herein, "erythroblast" means a nucleated precursor cell fromwhich a reticulocyte develops into an erythrocyte. "Normoblast" means anucleated red blood cell, the immediate precursor of an erythrocyte.

As used herein, "embryo" means a cell or cells from conception throughthe second month of gestation. Typically the developmental stages afterthe embryonic stage until birth are designated as fetal.

Fetal blood cells are rare cells circulating in the maternal bloodstream. Fetal cells are believed to "leak" into the maternal bloodstream through the placenta. Estimates of the frequency of this rareevent vary, but have been reported as approximately 1 in 10⁸ to 1 in10¹¹ cells. Holzgreve, W. et al., Lancet (1990) i:1220. During the earlyperiod of gestation, fetal red blood cells may be nucleated. Thus,unlike non-nucleated fetal erythrocytes, they contain fetal DNA and canbe used for genetic analysis of the fetus without the necessity ofinvasive procedures.

Ontogeny of Hemoglobin

Approximately 99% of adult hemoglobin is composed of two alpha chainsand two beta chains with about 1% comprising two alpha chains and twodelta chains. Fetal hemoglobin contains two alpha and two gamma chains.

Three earlier embryonic hemoglobins are constructed with zeta(alpha-like) and epsilon (beta-like chains). Embryonic Gower 2 is madeup to two alpha chains and two epsilon chains, while embryonic Gower 1hemoglobin is made up of two zeta chains and two epsilon chains, andembryonic hemoglobin Portland consists of two zeta chains and two gammachains. See Gale, et al., Nature, 280:162 (1979); and Maniatis, et al,Ann. Rev. Genetics, 14:145 (1980).

Through the present invention it has been demonstrated that embryonicglobin chains are still present in fetal RBCs up to about 22 menstrualweeks of gestation, although the messenger RNA is no longer present.Preferably, these chains are detected from about 9 to about 20 menstrualweeks. The fetus eventually switches to the production of fetalhemoglobin, which is found as approximately 1% of the hemoglobin in anadult. There are no embryonic hemoglobins in the adult RBCs.

Hemoglobin Antibodies

The various specific hemoglobins in RBCs can be distinguished usingantibodies or antibody fragments specific to the antigenic sites of theglobin chain. Antibodies to adult globins (alpha and beta), to fetalglobin (gamma) and to embryonic epsilon globin are commerciallyavailable. Accurate Chemical and Scientific Corporation (Westbury,N.Y.), and Cortex Biochem (San Leandro, Calif.) supply the epsilonglobin antibody.

A number of immunogens can be used to produce antibodies specificallyreactive with hemoglobin chain proteins. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Naturally occurring protein also can be used either in pureor impure form. Synthetic peptides made using the hemoglobin chainprotein amino acid sequence also can be used as an immunogen for theproduction of antibodies to the proteins. Recombinant protein can beexpressed in eukaryotic or prokaryotic cells, and purified. The productis then injected into an animal capable of producing antibodies.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purified protein,is mixed with an adjuvant and animals are immunized. The animal's immuneresponse to the immunogen preparation is monitored by taking test bleedsand determining the titer of the reactivity. When appropriately hightiters of antibody to the immunogen are obtained, blood is collectedfrom the animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the protein can be done ifdesired. See Harlow, et al, Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York (1988).

Monoclonal antibodies can be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell. See Kohler, et al., Eur. J. Immunol., 6:511-519 (1976).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells can be enhanced by various techniques, includinginjection into the peritoneal cavity of the vertebrate host.Alternatively, one can isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse, etal., Science, 246:1275-1281 (1989).

Various components or fragments of antibodies can be used in the presentinvention. The variable regions of an immunoglobulin are the portionsthat provide antigen recognition specificity. In particular, thespecificity resides in the complementary determining regions (CDRs),also known as hypervariable regions, of the immunoglobulins. Theimmunoglobulins may exist in a variety of forms including, for example,Fv, Fab, F(ab'), F(ab')₂, and other fragments, as well as single chains.See Huston, et al., Proc. Nat. Acad. Sci. U.S.A., 85:5879-5883 (1988)and Bird, et al., Science 242:423-426 (1988). See, generally, Hood, etal., Immunology, Benjamin, N. Y., 2nd ed. (1984), and Hunkapiller andHood, Nature, 323:15-16 (1986). Single-chain antibodies, in which genesfor a heavy chain and a light chain are combined into a single codingsequence, can also be used. Immunoglobulin polypeptide also encompassesa truncated immunoglobulin chain, for example, a chain containing lessconstant region domains than in the native polypeptide. Such truncatedpolypeptides can be produced by standard methods such as introducing astop codon into the gene sequence 5' of the domain sequences to bedeleted. The truncated polypeptides can then be assembled into truncatedantibodies. Antibodies as used herein also include bispecific antibodieswhich can be produced such as by the methods described in the followingreferences: Glennie et al., J. Immunol., 139:2367-2375 (1987); Segal, etal., Biologic Therapy of Cancer Therapy of Cancer Updates, 2(4):1-12(1992); and Shalaby, et al., J. Exp. Med., 175:217-225 (1992).Monospecific and bispecific immunoglobulins can also be produced byrecombinant techniques in prokaryotic or eukaryotic host cells.

"Chimeric" antibodies are encoded by immunoglobulin genes that have beengenetically engineered so that the light and heavy chain genes arecomposed of immunoglobulin gene segments belonging to different species.For example, the variable (V) segments of the genes from a mousemonoclonal antibody may be joined to human constant (C) segments. Such achimeric antibody is likely to be less antigenic to a human thanantibodies with mouse constant regions as well as mouse variableregions.

As used herein, the refers to an antibody also refers to an antibodythat includes an immunoglobulin that has a human-like framework and inwhich any constant region present has at least about 85-90%, andpreferably about 95% polypeptide sequence identity to a humanimmunoglobulin constant region, a so-called "humanized" immunoglobulin.See for example, PCT Publication WO 90/07861. Hence, all parts of such a"humanized" immunoglobulin, except possibly the complementarydetermining regions (CDRs), are substantially identical to correspondingparts of one or more native human immunoglobulin sequences. Wherenecessary, framework residues also can be replaced with those within oracross species especially if certain framework residues are found toaffect the structure of the CDRs. A chimeric antibody can also containtruncated variable or constant regions.

The term "framework region", as used herein, refers to those portions ofimmunoglobulin light and heavy chain variable regions that arerelatively conserved (i.e., other than the CDRs) among differentimmunoglobulins in a single species, as defined by Kabat, et al.,Sequences of Proteins of Immunologic Interest, 4th Ed., U.S. Dept.Health and Human Services (1987). As used herein, a "human-likeframework region" is a framework region that in each existing chaincomprises at least about 70 or more amino acid residues, typically 75 to85 or more residues, identical to those in a human immunoglobulin.

Human constant region DNA sequences can be isolated in accordance withwell known procedures from a variety of human cells, but preferably fromimmortalized B-cells. The variable regions or CDRs for producing thechimeric immunoglobulins of the present invention can be similarlyderived from monoclonal antibodies capable of binding to the embryonichemoglobins or their chains and will be produced in any convenientmammalian system, including, mice, rats, rabbits, human cell lines, orother vertebrates capable of producing antibodies by well known methods.Variable regions or CDRs may be produced synthetically, by standardrecombinant methods including polymerase chain reaction (PCR) or throughphage-display libraries. For phage display methods, see for example,McCafferty, et al., Nature, 348:552-554 (1990); Clackson, et al.,Nature, 352:624-628; and Marks, et al., Biotechnology, 11:1145-1149(1993). Suitable prokaryotic systems such as bacteria, yeast and phagecan be employed.

Suitable source cells for the DNA sequences and host cells forimmunoglobulin expression and secretion can be obtained from a number ofsources, such as the American Type Culture Collection ("Catalogue ofCell Lines and Hybridomas," Fifth edition (1985) Rockville, Md.,U.S.A.).

In addition to the chimeric and "humanized" immunoglobulins specificallydescribed herein, other substantially identical modified immunoglobulinscan be readily designed and manufactured utilizing various recombinantDNA techniques well known to those skilled in the art. In general,modifications of the genes can be readily accomplished by a variety ofwell-known techniques, such as PCR and site-directed mutagenesis. SeeGillman and Smith, Gene, 8:81-97 (1979) and Roberts, et al., Nature,328:731-734 (1987).

Alternatively, polypeptide fragments comprising only a portion of theprimary immunoglobulin structure can be produced. For example, it may bedesirable to produce immunoglobulin polypeptide fragments that possessone or more immunoglobulin activities in addition to, or other than,antigen recognition (e.g., complement fixation).

Labels

The antibodies or fragments can be labeled directly or indirectly fortheir isolation and identification. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers,chemiluminescers, magnetic particles, haptens, dyes, and the like. SeeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also included are reporter groups, such asbiotin, which bind to groups such as streptavidin or avidin, which inturn are bound directly or indirectly to enzymes, such as alkalinephosphatase or horseradish peroxidase. Fluorescers or fluorochromesinclude fluorescein, coumarin, rhodamine, phycoerythrin, sulforhodamineacid chloride (Texas red), and the like.

These detectable labels have been well-developed in the field of theinvention and, in general, most any label can be applied. Preferably,enzymes or fluorescers are utilized.

Blood Sampling

The method of the present invention preferably utilizes a blood samplefrom the mother during gestation; however, other sources of fetal cellscan be used besides those isolated or identified in the maternalcirculation. Fetal tissue can be obtained via amniocentesis, CVS,fetoscopy or cordocentesis for analysis as described above.

In those instances wherein maternal blood is the source of fetal cells,the blood sample can be whole blood or a fractionated component of theblood containing the fetal erythrocyte or erythroblast cells of choicein a mononuclear cell layer. Typically, the blood source is prepared toenrich the concentration of fetal cells before and/or after the use ofthe anti-embryonic hemoglobin antibody of the claimed method. Further,the blood sample can be suspended, air-dried, or chemically-fixed on toa solid matrix before contact with the antibody.

Although any maternal or fetal mammal can be the source of fetal tissue,the preferred source is human. Other domestic mammals are alsopreferred, such as dogs, cats, cows horses and the like.

Enrichment Methods Density Gradients

In addition to blood fractionation, methods for the isolation orenrichment of blood cells have been described which use densitygradients containing cell aggregating or clumping agents such asmethylcellulose, Isopaque™, dextran and Ficoll™, as described in Boyum,Scand. J. Clin. Lab. Invest., 21 (Suppl. 97):31-50 (1968), and in Bhat,N. M. J. Immunol. Meth, 158:277-280 (1993). Isopaque™ is a sodiumN-methyl-3,5,-diacetamino-2,4,6-triiodobenzoate, as described in Boyum.Ficoll™ (Accurate Chemical and Scientific Corporation, Westbury N.Y.) isa synthetic high polymer made by the copolymerization of sucrose andepichlorohydrin. The molecules have a branched structure with a highcontent of hydroxyl groups giving solubility in aqueous media. Many ofthese agents are freely diffusible. These agents cause erythrocyteclumping, and thus provide methods for isolating leukocytes from redblood cells. However, under these cell-aggregating conditions, fetalnucleated red blood cells may become physically trapped within a clumpof aggregated maternal red blood cells, and therefore will sediment withmaternal erythrocytes, as the average density of the clump determinesits sedimentation characteristics.

Percoll density gradients have been described in Rennie, et al ClinicaChemica Acta, 98:119-125 (1979), and in Vincent and Nadeau, Anal.Biochem., 141:322-328 (1984). In the Rennie study, an isotonic Percolldensity gradient was used to age-fractionate erythrocytes. Leukocytes(white blood cells) were removed prior to the centrifugation process, asthey co-fractionated with erythrocytes in isotonic gradient conditions.

Ganshert-Ahlert, et al, Am. J. Obstet. Gynecol., 1350-1355 (1992) andPCT Publication WO 93/23754, describe a method of enriching for fetalnucleated erythrocytes using a triple density gradient on whole maternalblood, followed by use of the transferrin receptor to enrich fetalnucleated red blood cells. A flow cytometry or magnetic separation stepis required to enrich the labelled cells. As noted in theGanshert-Ahlert reference, the use of the transferrin receptor stilldoes not provide a reliable identification of fetal cells in acirculating maternal cell population.

A preferred enrichment method for isolating fetal nucleated red bloodcells from a maternal population comprises the steps of centrifuging theblood sample in a first centrifugation vessel to obtain a red blood cellfraction; transferring the red blood cell fraction to an upper portionof a second centrifugation vessel, the second centrifugation vesselhaving a density gradient medium consisting of a colloid dispersed in ameltable gel, wherein the colloid is capable of maintaining the redblood cells in a substantially unaggregated state; hemolyzing maternalerythrocytes in the red blood cell fraction to obtain an enriched fetalerythrocyte fraction; melting the gel; and centrifuging the enrichedfetal erythrocyte fraction through the density gradient medium to obtaina fraction enriched in fetal nucleated erythrocytes. See U.S. Pat. No.5,432,054.

The first centrifuge step provides an initial enrichment which separatesthe low density nucleated red blood cell fraction and all the whiteblood cells from the more dense un-nucleated red blood cells, and fromthe serum, and serum proteins. Preferably, the first centrifuge tube ismade of soft plastic, in order to facilitate the movement of the bloodcells through the tube. Suitable tubes are described in U.S. Pat. No.5,422,018. Plastic hourglass shaped tubes are preferably supportedwithin the centrifuge, to prevent excessive deformity or collapse of thetube at the narrow central channel portions. Support may be provided byany suitable means. For example, a solid removable support cast may bewrapped around the tube. In a preferred embodiment, the tube issupported in a liquid support medium within a larger vessel, such as atest tube. The level of liquid is at least high enough to cover thenarrow portion of the tube. Preferably, the weight of the volume of theliquid support medium displaced by the sample tube is approximatelyequivalent to the weight of the volume of the sample tube and itscontents. A preferred liquid support medium for use is water.

After the first centrifugation step, a fraction containing the nucleatedred blood cells is obtained. This fraction also includes the white bloodcells. The top of the tube contains the plasma fraction. The nucleatedred blood cells, which are more dense than plasma but less dense thanother red blood cells, will fractionate at the top of the red blood cellstack found just below the plasma and will be variably mixed with whiteblood cells. The use of a precalibrated first centrifuge tube permitseasy extraction of the relevant fraction from the narrow portion of thefirst tube, thus minimizing inclusion of other blood fractions,including serum and plasma from the first centrifugation step.

The fraction containing the red blood cells and white blood cells can behemolyzed to differentially disrupt the maternal red blood cells.Differential hemolysis of the maternal red blood cells permits thedestruction of a significant number of the remaining maternal red bloodcells while preserving the majority of the fetal-origin cells. SeeBoyer, et al, Blood, 47(6): 883-897 (1976). The differential hemolysismay occur in any suitable reaction vessel. In a preferred embodiment,the differential hemolysis of the maternal red blood cells occurs in anupper portion of the second centrifugation vessel, such that thehemolysis reaction may be stopped by centrifuging the reaction products,i.e. the preserved red blood cells, into the density gradient medium,thus removing the red blood cells from the hemolysis reagents.

The differential hemolysis utilizes the fact that red blood cells may bedisrupted in solutions containing hemolyzing agents such as ammonium(NH₄ --) and bicarbonate (HCO₃ --) ions. The cell disruption may bedecelerated by inhibitors of the enzyme carbonic anhydrase. Carbonicanhydrase levels are at least five fold higher in adult erythrocytesthan in fetal erythrocytes. Thus, the rate of NH₄ --HCO₃ mediatedhemolysis is slower for fetal red blood cells, including fetal nucleatedred blood cells, than for adult red blood cells, particularly in thepresence of carbonic anhydrase inhibitors. Preferred carbonic anhydraseinhibitors for use in the invention include acetazolamide, ethoxzolamide(6-ethoxyzolamide, Sigma Chemical Co.) and methoxzolamide.

Differential hemolysis results in a population of white blood cellstogether with red blood cells enriched for fetal red blood cells. Theenriched fetal red blood cell fraction is then centrifuged through thedensity gradient medium in order to harvest the fraction enriched forfetal nucleated red blood cells, and to remove red blood cell fragmentsresulting from the hemolysis reaction and the majority of white bloodcells. The fetal nucleated red blood cells present in an initial sampleof 20 ml of peripheral blood may be reduced into a 2 microliter sample,thus providing easy identification and analysis on a microscope slide,or by polymerase chain reaction.

The second centrifugation step utilizes a density gradient medium. Afterhemolysis, the nucleated red blood cells are expected to equilibrate ina density gradient at approximately the same density as granulocytes, acomponent of the white blood cell fraction, as described in PCTInternational Application No. WO 93/23754. The tonicity and density ofthe gradient medium can allow separation and enrichment of the fetalnucleated erythrocytes from the white blood cell components of thesample.

The preferred density gradient medium is comprised of a colloiddispersed in a meltable gel. See U.S. Pat. No. 5,489,386. The colloidimparts the required density to the gradient medium. Thus, by alteringthe concentration of the colloid, the density of the medium may becorrespondingly altered. The particulate nature of the colloid enablesimmobilization of separate layers of density without diffusion of onelayer into another while in the gel state. Further, the colloid iscapable of maintaining the blood cells in a substantially unaggregatedstate. A preferred colloid which imparts the density to the medium ispolyvinyl-pyrrolidone coated silica, for example, Percoll™, manufacturedby Pharmacia, and available from Sigma Chemical Co.

The density gradient medium for use in enriching fetal nucleatederythrocytes is hypertonic. Under hypertonic conditions, red blood cellsshrink and thus become more dense. Under these conditions, white bloodcells maintain a constant density. Thus, by selectively shrinking theerythrocytes in a hypertonic medium, the density of these cellsincreases and they equilibrate within the gradient at a differentdensity from the white blood cells.

Enrichment Methods--Flow Cytometry and Others

Enrichment of the blood sample can be accomplished using othertechniques, such as cell panning, microdissection using lightmicroscopy, flow cytometry, and/or magnetic bead or particle separation.For example, antibodies specific to maternal or mature cell markers,and/or antibodies specific to a second fetal marker can be added to themethod of the present invention to further enrich the sample for fetalcells. Either a positive or negative selection approach can be applied,i.e., enhancing the desired fetal cells or eliminating the unwantedmature cells. The use of an antibody-bound column can be used alone orin conjunction with other enrichment techniques.

Mueller, et al, in Lancet, 336:197 (1990) described a method ofisolating placenta-derived trophoblast cells in the blood of pregnantwomen using magnetic beads. Other variations in methods for conjugatingantibodies to beads also exist. See Thomas, et al., J. Immunol., 120:221(1989) and deKretser, et al., Tissue Antigens, 16:317 (1980). Analternative method of enrichment was discussed in Berenson, et al., J.Immunol. Methods, 91:11 (1986) in which the high affinity between theprotein avidin and the vitamin biotin was exploited to create anindirect immunoadsorptive procedure.

In flow cytometry, cells can be analyzed and sorted on a flow sorterbased on the properties of the cells to scatter light forward and to theside. In each experiment parameters are empirically establishedregarding the forward and side scatter properties. In general, the gainon the photomultiplier tubes detecting the forward-scatter light and theside-scattered light is adjusted in each dimension to distribute thearray of signals from the cells across the channels available foranalysis in a manner well known to one skilled in the art. Under thesecircumstances a characteristic pattern, or scattergram, is observed.Analysis of blood samples reveals three major cell types in thescattergram, namely, monocytic cells, lymphocytes and granulocytes, eachof which has distinguishable light scattering characteristics. Themonocytic cell region, the granulocytic cell region and the lymphocyticcell region of the scattergram are gated so that cells which areclassified as monocytes, granulocytes or lymphocytes can be analyzedfurther or collected by flow sorting.

Further analysis can be carried out by staining the cells withflorescent-coupled monoclonal antibodies or by subjecting the cells toin situ hybridization with fluorescent-coupled oligonucleotide ornucleic acid probes. Under these conditions cells that have particularlight scattering properties are also analyzed for the presence offluorescence. When fluorescent-coupled antibodies are used, controlexperiments are performed using isotypically matched control monoclonalantibodies. When fluorescent-coupled oligonucleotide probes are used,controls consist of oligonucleotide sequences unrelated to mammaliansequences.

Collected samples are deposited on one or more slides, with no more than2-3,000,000 cells deposited on any single slide; care is taken thatdeposited cells form a monolayer such that the concentration of cells onthe slide is low enough so that the cells do not overlap one another. Atother times, the cells are collected into microfuge tubes and fixed insuspension as described elsewhere.

A Coulter, Profile II, flow cytometer (Coulter, Haileah, Fla.) can beused to detect nucleic acids within fetal cells. An Epics Elite,(Coulter, Haileah, Fla.) system can be used to sort fetal cells from aspecimen of maternal blood.

Preferably, a fluorescence activated cell sorter (FACS) is used toperform flow cytometry and to identify the fetal cells, usingfluorescein as the label or dye directly or indirectly bound to theantibody.

Other Markers

A second fetal marker can be used to define the cell as fetal. Forexample, antibodies to representative fetal cell markers can be used.For fetal red blood cells, a fetal hemoglobin marker, such as anantibody to the fetal gamma chain of hemoglobin, is preferable.

Fetal-cell-specific RNA sequences also can be used as fetal cellmarkers. Such sequences are transcripts of, e.g., the fetal hemoglobingene. The sequences of these genes and others may be obtained from theGenetic Sequence Data Bank, GenBank, version 69.0. A DNA probe, orpopulation of probes, embodying any of these sequences is synthesized asan oligodeoxynucleotide using a commercial DNA synthesizer such as Model380B from Applied Biosystems, Inc., Foster City, Calif. Probes can becomprised of the natural nucleotide bases or know analogues of thenatural nucleotide bases, including those modified to bind labelingmoieties.

For negative selection, a mature cell marker can be used, such as anantibody to anti-CD45, anti-CD13 and/or anti-CD34, which selectivelybinds to white blood cells. Anti-CD44 antibodies can be included toremove contaminating maternal red blood cells. The addition of anti-CD31antibodies can specifically remove contaminating platelets. Preferably,antibodies directed to the adult beta chain of hemoglobin can beutilized.

Fetal Nucleic Acids and Proteins

Once the fetal cells are isolated from the maternal blood, they can becultured to increase the numbers of cells available for diagnosis. SeeFibach, et al., Blood, 73:100 (1989).

Particular fetal proteins and/or nucleic acids can be selected orisolated as follows. The cells can be lysed and thereby rendering thenucleic acid or protein available for analysis. In some instances, fetalDNA can be extracted from other complexes, e.g., by heat, making itaccessible for hybridization with nucleic acid probes. Prior toanalysis, the fetal DNA can be amplified via methods such as polymerasechain reaction (PCR) or ligase chain reaction (LCR).

If amplification is to be carried out, the sorted samples are amplifiedfor an appropriate number of cycles of denaturation and annealing (e.g.,approximately 25-60). Control samples can include a tube without addedDNA to monitor for false positive amplification. With propermodification of PCR conditions, more than one separate fetal gene can beamplified simultaneously. This technique, know as "multiplex"amplification, has been used with six sets of primers in the diagnosisof DMD. See Chamberlin, et al., Prenat. Diagn., 9:349-355 (1989). Whenamplification is carried out, the resulting amplification product is amixture that contains amplified fetal DNA of interest, i.e., the DNAwhose occurrence is to be detected and/or quantitated.

The amplified fetal DNA of interest and other DNA sequences areseparated, using known techniques. Subsequent analysis of amplified DNAalso can be carried out using known techniques, such as: digestion withrestriction endonuclease, ultraviolet light visualization of ethidiumbromide stained agarose gels, DNA sequencing, or hybridization withallele specific oligonucleotide probes. See Saiki, et al., Am. J. Hum.Genet., 43 (Suppl.):A35 (1988). Such analysis will determine whetherpolymorphic differences exist between the amplified "maternal" and"fetal" samples. The amplification mixture can be separated on the basisof size and the resulting size-separated fetal DNA is contacted with anappropriate selected DNA probe or probes (DNA sufficiently complementaryto the fetal DNA of interest that it hybridizes to the fetal DNA ofinterest under the conditions used). Generally, the DNA probes arelabelled using labels as described above.

After the size-separated fetal DNA and the selected DNA probes have beenmaintained for sufficient time under appropriate conditions forhybridization of complementary DNA sequences to occur, resulting inproduction of fetal DNA/DNA probe complexes, detection of the complexesis carried out using known methods. For example, if the probe islabelled, the fetal DNA/labelled DNA probe complex is detected and/orquantitated (e.g., by autoradiography, detection of the fluorescentlabel). The quantity of labelled complex (and, thus, of fetal DNA can bedetermined by comparison with a standard curve (i.e., a predeterminedrelationship between quantity of label detected and a given reading).

Detection of Genetic Abnormalities

The occurrence of fetal DNA associated with diseases or conditions canbe detected and/or quantitated by the present method. In each case, anappropriate probe is used to detect the sequence of interest. Forexample, sequences from probes St14 (Oberle, et al, New Engl. J. Med.,312:682-686 (1985)), 49a (Geurin, et al., Nucleic Acids Res., 16:7759(1988)), KM-19 (Gasparini, et al., Prenat. Diagnosis, 9:349-355 (1989)),or the deletion-prone exons for the Duchenne muscular dystrophy (DMD)gene (Chamberlain, et al., Nucleic Acids Res., 16:11141-11156 (1988))are used as probes. St14 is a highly polymorphic sequence isolated fromthe long arm of the X chromosome that has potential usefulness indistinguishing female DNA from maternal DNA. It maps near the gene forFactor VIII:C and, thus, can also be utilized for prenatal diagnosis ofHemophilia A. Primers corresponding to sequences flanking the six mostcommonly deleted exons in the DMD gene, which have been successfullyused to diagnose DMD by PCR, can also be used. See Chamberlain, et al.,Nucleic Acids Res., 16:11141-11156 (1988). Other conditions which can bediagnosed by the present method include Down's Syndrome, β-thalassemia(Cai, et al., Blood, 73:372-374 (1989); Cai, et al., Am. J. Hum. Genet.,45:112-114 (1989); Saiki, et al., New Engl. J. Med., 319:537-541(1988)), sickle cell anemia (Saiki, et al., New. Engl. J. Med.,319:537-541 (1988)), phenylketonuria (DiLella, et al., Lancet, 1:497-499(1988)) and Gaucher's disease (Theophilus, et al., Am. J. Hum. Genet.,45:212-215 (1989)).

The genetic abnormalities detected by the present invention can bedeletions, additions, amplifications, translocations or rearrangements.

For example, a deletion can be identified by detecting the absence ofhybridizable binding of the probe to a target sequence. To detect adeletion of a genetic sequence, a population of probes are prepared thatare complementary to the nucleic acid sequence that is present in anormal fetal cell but absent in an abnormal one. If the probes hybridizeto the sequence in the cell being tested, then the sequence is detectedand the cell is normal as to that sequence. If the probes fail tohybridize to cellular nucleic acid, then the sequence is not detected inthat cell and the cell is designated as abnormal, provided that acontrol sequence, such as the X chromosome, is detected in the samecell.

An addition can be identified by detecting binding of a labeled probe toa polynucleotide repeat segment of a chromosome. To detect an additionof a genetic sequence, such as an insertion in a chromosome or akaryotypic abnormality such as the trisomy of Chromosome 21 whichindicates Down's Syndrome, a population of probes are prepared that arecomplementary to the genetic sequence in question. Continuing with theDown's Syndrome example, if the probes complementary to Chromosome 21hybridize to three appearances of the Chromosome 21 sequence in thecell, then three occurrences of the Chromosome 21 sequence will bedetected and indicate the Down's Syndrome trisomic condition. If thedetection means is a fluorescent dye, for example, then three distinctpoints of fluorescence visible in each cell will indicate the trisomycondition.

When an amplification of a particular DNA fragment is present, there isan increase in the intensity of the signal from a labeled probe for thesequence which is subject to amplification. Using any number of imageanalysis systems, this signal is quantified and compared to normalcontrols to determine whether or not a particular amplification mutationis present.

A translocation or rearrangement can be identified by several methods.For example, a labeled first probe may be bound to a marker region of achromosome that has not translocated. A labeled second probe is thenbound to a second region of the same chromosome (for a rearrangement) ora second chromosome (for a translocation) and subsequently binding ofthe first and second probes is detected. Alternatively, a translocationcan be identified by first binding a labeled probe to a marker region ofa polynucleotide section of a chromosome that translocates orrearranges, usually during metaphase. Subsequently, binding of thelabeled probe is detected.

For example, to detect a translocation, a marker for the chromosome inquestion is identified, and a population of probes are prepared thatselectively hybridize to it. They are marked with a detectable label,such as a dye that fluoresces at a particular wavelength. The sequencethat translocates or rearranges in the abnormality being tested for isalso identified, and second population of probes are prepared thatidentify it. The members of a second population of probes are markedwith a distinguishably different label, such as a dye that fluoresces ata different wavelength from the first series of labeled probes. In situhybridization is performed using both populations of probes, and theresults of hybridization by each of the probe populations are compared.If the first and second labels are coincident on virtually all cellsamples, no translocation has taken place. If the first label is foundnot to coincide with the second label on a significant fraction ofsamples, then a translocation or rearrangement has taken place. SeeSpeleman, Clinical Genetics, 41(4):169-174 (1992); and Gray, Progress inClinical and Biol. Res., 372:399-411 (1991).

Nucleic Acid Hybridization

The term "nucleic acids", as used herein, refers to either DNA or RNA."Nucleic acid sequence" or "polynucleotide sequence" refers to a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotidebases read from the 5' to the 3' end. It includes both self-replicatingplasmids, infectious polymers of DNA or RNA and nonfunctional DNA orRNA.

"Nucleic acid probes" may be DNA or RNA fragments. DNA fragments can beprepared, for example, by digesting plasmid DNA, or by use of PCR, orsynthesized by either the phosphoramidite method described by Beaucageand Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by thetriester method according to Matteucci, et al., J. Am. Chem. Soc.,103:3185 (1981). A double stranded fragment may then be obtained, ifdesired, by annealing the chemically synthesized single strands togetherunder appropriate conditions or by synthesizing the complementary strandusing DNA polymerase with an appropriate primer sequence. Where aspecific sequence for a nucleic acid probe is given, it is understoodthat the complementary strand is also identified and included. Thecomplementary strand will work equally well in situations where thetarget is a double-stranded nucleic acid.

The phrase "selectively hybridizing to" refers to a nucleic acid probethat hybridizes, duplexes or binds only to a particular target DNA orRNA sequence when the target sequences are present in a preparation oftotal cellular DNA or RNA. "Complementary" or "target" nucleic acidsequences refer to those nucleic acid sequences which selectivelyhybridize to a nucleic acid probe. Proper annealing conditions depend,for example, upon a probe's length, base composition, and the number ofmismatches and their position on the probe, and must often be determinedempirically. For discussions of nucleic acid probe design and annealingconditions, see, for example, Sambrook, et al., Molecular Cloning: ALaboratory Manual, (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory,(1989) and Ausubel, et al., Current Protocols in Molecular Biology, ed.Greene Publishing and Wiley-Interscience, New York (1987).

Nucleic acids for use as probes are chemically synthesized according tothe solid phase phosphoramidite triester method first described byBeaucage and Carruthers, Tetrahedron Lett., 22(20):1859-1862 (1981)using an automated synthesizer, as described in Needham-VanDevanter, etal., Nucleic Acids Res., 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson and Regnier, J. Chrom.,255:137-149 (1983). The sequence of the synthetic oligonucleotide can beverified using the chemical degradation method of Maxam and Gilbert, inGrossman and Moldave, eds. Academic Press, New York, Methods inEnzymology, 65:499-560 (1980).

A variety of methods for specific DNA and RNA measurement using nucleicacid hybridiziation techniques are known to those of skill in the art.For example, one method for evaluating the presence or absence of DNA ina sample involves a Southern transfer. Briefly, the digested genomic DNAis run on agarose slab gels in buffer and transferred to membranes.Hybridization is carried out using the nucleic acid probes. Preferablynucleic acid probes are 20 bases or longer in length. (See Sambrook, etal. for methods of selecting nucleic acid probe sequences for use innucleic acid hybridization.) Visualization of the hybridized portionsallows the qualitative determination of the presence or absence of DNA.

Similarly a Northern transfer may be used for the detection of mRNA. Inbrief, the mRNA is isolated from a given cell sample using an acidguanidinium-phenol-chloroform extraction method. The mRNA is thenelectrophoresed to separate the mRNA species and the mRNA is transferredfrom the gel to a nitrocellulose membrane. As with the Southern blots,labeled probes are used to identify the presence or absence ofappropriate mRNA.

A variety of nucleic acid hybridization formats are known to thoseskilled in the art. For example, common formats include sandwich assaysand competition or displacement assays. Hybridization techniques aregenerally described in "Nucleic Acid Hybridiziation, A PracticalApproach," Ed. Hames, and Higgins, IRL Press, 1985; Gall and Pardue,Proc. Natl. Acad. Sci., USA, 63:378-383 (1969); and John, Burnsteil andJones, Nature, 223:582-587 (1969).

Ethanol, e.g., 80% ethanol/water (v/v), is desirably used as a fixativeduring preparation of the cells for in situ hybridization. Other usefulprecipitation fixatives include acetic acid, methanol, acetone, andcombinations thereof, for example, ethanol/methanol mixture 3:1. Otheruseful fixatives will be known to one skilled in the art. Fixatives andhybridization of fixed cells, in general, are discussed in U.S. Pat. No.5,225,326. Fixatives should provide good preservation of cellularmorphology, should preserve and maintain accessibility of antigens, andpromote a high hybridization efficiency. Some salts and extremetemperature, such as waving a slide over a flame, may also function asfixatives.

The fixative can contain a compound which fixes the cellular componentsby cross-linking these materials together, for example,paraformaldehyde, glutaraldehyde or formaldehyde. Cross-linking agents,while preserving ultrastructure, often reduce hybridization efficiencyby forming networks trapping nucleic acids and antigens and renderingthem inaccessible to probes and antibodies. Some cross-linking agentsalso covalently modify nucleic acids, preventing later hybrid formation.

The hybridization solution typically comprises a chaotropic denaturingagents include formamide, urea, thiocyantem guanidine,thrichloroacetate, tetramethyamine, perchlorate, and sodium iodide. Anybuffer which maintains pH at least between about 6.0 and about 8.0 andpreferably between 7.0 and 8.0 can be utilized.

Miscellaneous

Many types of solid supports can be used to practice the invention.Supports include glass, nylon, nitrocellulose and the like. Mostpreferably, glass or plastic microscope slides are use. The use of thesesupports and the procedures for depositing specimens thereon is known tothose of skill in the art. The choice of support material will dependupon the procedure for visualization of cells and the quantitationprocedure used.

In addition, nuclear stains can be used to recognize chromatin, nuclearproteins, nuclear components, DNA and the like. These stains includemethylene blue, hematoxylin, DAP 1, propidinium iodide, thionin and thelike.

Various chromosomal staining techniques also are encompassed by thepresent invention. Chromosomal painting is generally described in U.S.Pat. No. 5,447,841. Typically the heterogeneous mixtures of labellednucleic acid fragments or probes are free from repetitive sequences. Theentire genome, single chromosomes, subregions of chromosomes, and thelike can be stained, usually during the metaphase cycle of the cell.

Kits

A kit for use in carrying out the present method of isolating anddetecting fetal DNA of interest, such as a chromosomal abnormalityassociated with a disease or other condition, in a maternal blood samplecan be produced. It includes, for example, a container for holding thereagents needed; the reagents and, optionally, a solid support for usein separating fetal nucleated cell/specific antibody complexes fromother sample components or for removing maternal cells complexed with aspecific antibody.

Preferably, provided by the invention is a kit for identifying a fetalnucleated erythrocyte or erythroblast cell comprising a labelledanti-embryonic hemoglobin antibody, wherein the label is a fluorochrome,enzyme, or biotin. Alternatively, the anti-embryonic hemoglobin antibodyis conjugated with an antigenic hapten and a labeled second antibodydirected to the hapten or hemoglobin antibody is used for capture. Otheringredients can be further components in the kit, such as a bloodfractionation tube, hemolysis reagents, density gradient medium, lysisagents, labelled nucleic acid probes, and the like along withinstructions for use.

For example, reagents in a kit to be used in detecting fetal DNA ofinterest after amplification of fetal DNA by PCR can include: 1) atleast one antibody specific for an embryonic hemoglobin or chain;selected DNA primers for use in amplifying fetal DNA by PCR; and atleast one DNA probe complementary to the fetal DNA to be detected (fetalDNA of interest). The kit, as indicated, can also include a solidsupport to be used in separating complexes formed from other samplescomponents. Such solid support can be, for example, a glass slide,nitrocellulose filter, or immunomagnetic beads and can have affixesthereto an antibody selective for the antibody present in the fetalnucleated cell/specific antibody complexes. Other kits can include asolution containing a fixation/hybridization cocktail and one or morelabeled probes. A kit also would provide means and instructions forperforming the hybridization reaction. A kit could also include aphotographic film or emulsion with which to record results of assayscarried out with the invention.

Yet another aspect of the present invention would be a kit to enrich anddetect fetal cells within a blood specimen, e.g., maternal or umbilicalcord blood. Such a kit may contain one or more reagents to prepare adensity gradient that concentrates fetal cells. Labeled antibodies todetect fetal cells and/or probes specific for fetal cell mRNA and/orDNA, and means and instructions for performing fetal cell enrichmentalso would be included.

An alternative kit can contain one or more antibodies, desirably boundto a solid support, to positively or negatively concentrate fetal cellswithin the specimen, probes specific for chromosome specific DNAsequences, means and instructions for performing fetal cell enrichmentusing density gradient centrifugation or flow cytometry, and optionallyone or more reagents to prepare a density gradient that concentratesfetal cells.

Such a kit would optionally provide reagents and materials for use in anautomated system for the performance of any of the methods of thepresent invention.

EXAMPLES

The following example is provided merely for the purposes ofillustration and are not to be construed in any way as limiting thescope of the present invention. Those skilled in the art will recognizethat certain variations and modifications can be practiced within thescope of the invention.

Wedge smears were prepared from 14-week gestation abortus cord blood,whole blood from the mother (post-abortion), and a 1:25 mixture of theabove cord blood into the maternal blood sample. The smears were fandried for about half hour at room temperature, fixed in 100% methanol at-20° C. for 10 minutes, and then in 100% acetone for 10 minutes at -20°C.

The slides were then washed in PBS (10 mM Phosphate Buffered Saline (pH7.4) for 5 minutes at room temperature with moderate agitation and fixedin 2% formaldehyde/PBS at room temperature for 10 minutes with moderateagitation. After washing the slides twice in PBS for 5 minutes at roomtemperature with moderate agitation, the slides were washed twice in TBS(100 mM Tris-HCl pH 7.6) for 5 minutes at room temperature with moderateagitation.

The specimens were treated with blocking agent by depositing 200 μm ofTNBB (0.5% blocking reagent (Boehringer Mannheim) and 1% BSA in 100 mMTBS) on a 24×60 coverslip for each side to be stained. The smears werethen placed sample-side down onto the spot of TNBB. The slide-coverslipwas placed coverslip-down in a plastic slide storage box, which in turnwas placed in an open moist chamber containing water-dampened papertowels. Finally, the moist chamber was placed in a desiccator and avacuum (Precision Vacuum Pump Model DD20) was applied to the entireconstruct for 15 minutes at room temperature. The slides were removedfrom the desiccator and immunostained.

The coverslips were gently removed and 100 μl of cocktail containing a1:250 dilution of mouse anti-HbE (embryonic epsilon hemoglobin)(monoclonal) and a 1:10 dilution of rabbit anti-HbF (fetal gamma globin)(polyclonal) biotinylated antibody in TNBB/0.75% Tween 20 was added toeach slide. The smear was covered with a fresh coverslip, and as above,the slide was incubated coverslip-down in a plastic slide storage box ina moist open chamber in a desiccator under vacuum for 15 minutes.

The coverslips were gently removed and the slides washed at roomtemperature with moderate agitation in TBS for 10 minutes, and thenwashed twice for 5 minutes. 100 μl of a second cocktail containing 1:100dilution of FITC conjugated goat anti-mouse antibody and 1:100 dilutionof Texas red conjugated horse anti-rabbit antibody in TNBB/0.75% Tween20 was added to each slide and incubated under vacuum, as above.

The coverslips were gently removed and the slides washed at roomtemperature with moderate agitation in TBS for 10 minutes, and thenwashed twice for 5 minutes. The slides were then air dried and mountedin Vectrashield/DAPI.

Result:

The above system stained embryonic hemoglobin (hBE, epsilon hemoglobin)with green FITC fluorescence, and fetal hemoglobin (HbF, gammahemoglobin) with Texas Red fluorescence. And addition, the nucleus ofnucleated cells were stained blue by the DAPI counterstain.

The anti-HbE antibody (embryonic hemoglobin) was very specific. FITCfluorescence (embryonic hemoglobin) was observed only in red blood cellsfrom the cord and cord:maternal mixture smears. Both nucleated andmature HbE-positive red blood cells were observed. No FITC-stained whiteblood cells were found in any of the smears.

Texas Red fluorescence (gamma hemoglobin) was observed in mature redblood cells in all three smear types: cord, maternal, and mixture. Itwas also observed in nucleated red blood cells from the cord andcord:maternal mixture smears. Nucleated red blood cells, stained orotherwise, were not found in the maternal smear. Texas Red fluorescenceis also observed in the cytoplasm of some granulocytes from cord andmaternal smears. Whether this red fluorescence in granulocytesrepresented hemoglobin uptake by the granulocytes, or was an artifact ofthe polyclonal orgins of the antibody, is not known.

The cord and mixture smears also contained nucleated and mature redblood cells that stained positive for both gamma and embryonichemoglobin.

All publications, patents, and patent applications herein areincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

The foregoing description of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise form disclosed, and many modifications andvariations are possible in light of the above teaching, and are intendedto be within the scope of the invention.

Although the foregoing invention has been described in some detail byway of illustration and example, for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method of identifying a fetal erythrocyte orerythroblast cell in a blood sample from a pregnant female from 9 to 22menstrual weeks, the method comprising:a) contacting the blood samplewith an antibody, or antibody fragment thereof, directed to an embryonicepsilon globin chain of hemoglobin, wherein the antibody or fragment isa first fetal marker and wherein the antibody or fragment will bind thefetal cell; and wherein the blood sample is air-dried orchemically-fixed on to a solid matrix before or after being contactedwith the antibody or fragment; and b) identifying the cells which bindto the antibody or fragment as fetal erythrocyte or erythroblast cells.2. The method of claim 1 wherein the blood sample is human maternalblood and wherein the fetal erythrocyte is a nucleated erythrocyte. 3.The method of claim 1 wherein the antibody is a polyclonal antibody. 4.The method of claim 1 wherein the antibody is a monoclonal antibody. 5.The method of claim 1 wherein the antibody is directly or indirectlylabelled.
 6. The method of claim 1 further comprising contacting theblood sample with a second fetal marker.
 7. The method of claim 6wherein the second fetal marker is an antibody, or antibody fragmentthereof, directed to the fetal gamma globin chain of hemoglobin.
 8. Themethod of claim 1 further comprising contacting the blood sample with amature cell marker.
 9. The method of claim 8 wherein the mature cellmarker is an antibody, or antibody fragment thereof, directed to theadult beta globin chain of hemgglobin.
 10. The method of claim 1 furthercomprising enriching the concentration of the fetal cells in the bloodsample before being contacted with the antibody or fragment, for theidentification of the fetal cells.
 11. The method of claim 10 whereinthe fetal cells are enriched through flow cytometry, bloodfractionation, density gradient separation, or magnetic bead separation.12. The method of claim 1 further comprising selecting or isolating anucleic acid or protein contained within the fetal cells after the fetalcells are identified.
 13. The method of claim 12 wherein the nucleicacid or protein is amplified or detected.
 14. A kit for identifying afetal nucleated erythrocyte or erythroblast cell in a blood sample froma pregnant female from 9 to 22 menstrual weeks comprising:a) a labeledanti-embryonic epsilon globin chain of hemoglobin antibody; and b)instructions for use.
 15. The kit of claim 14 wherein the antibody islabelled with a fluorochrome or an enzyme.
 16. A kit for identifying afetal nucleated erthyrocyte or erythroblast cell in a blood sample froma pregnant female from 9 to 22 menstrual weeks comprising:a) ananti-embryonic epsilon globin chain of hemoglobin antibody labeled withan antigenic hapten; b) a labelled second antibody directed to thehapten or to the anti-embryonic epsilon globin chain of hemoglobinantibody; and c) instructions for use.
 17. A kit for identifying a fetalnucleated erythrocyte or erythroblast cell in a blood sample from apregnant female from 9 to 22 menstrual weeks comprising:a) ananti-embryonic epsilon globin chain of hemoglobin antibody conjugated tobiotin; b) a labeled avidin or streptavidin molecule; and c)instructions for use.
 18. A kit for selecting or isolating a nucleicacid sequence within an identified fetal nucleated erythrocyte orerythroblast cell in a blood sample from a pregnant female from 9 to 22menstrual weeks comprising:a) a labeled anti-embryonic epsilon globinchain of hemoglobin antibody used to identify the fetal cells; b) adensity gradient medium; c) an agent for lysing the fetal cells aftersaid fetal cells are identified; d) a labeled nucleic acid probespecific for the selected or isolated nucleic acid sequence; and e)instructions for use.
 19. The kit of claim 18 wherein the labellednucleic acid probe is a DNA or RNA probe.